Heparin derivatives and methods of making same



United States Patent "ice 3,207,665

Patented Sept. 21, 1965 3,207,665 (1 1,3 ,3-tetramethyl-butyl-phenoxy)-ethoxy] -ethyl am- HEPARIN DERIVATEVES AND METHODS OF monium chloridewhich is also known under the trade- M AKING SAME mark Hyamine 1622,trimethyl-(methyldodecylbenzyl)- Robert Bucourt, Clichy-sous-Bois,Seine-et-Oise, France, ammonium chloride, dilauryldimethyl ammoniumch10- assignor to Roussel-UCLAF, Paris, France, a corporaride and othersuitable compounds. tion of France A preferred mode of the process ofthe invention com- NO P Filed J -l m- Q- 333,027 prises reacting ethylchloroformate with a high molecular Claims priority, appltgizttzioollsgrg 21, weight long chain quaternary ammonium salt of heparine 11 Claims.2 in the presence of an inert solvent such as tetrahydrofuran to formthe corresponding mixed anhydride, reacting the The invention relates tothe novel compound, N-benzyl latter with monobenzylamine at temperaturesabout 0 heparinamide and its alkali metal salts and to a novel C. toform the corresponding quaternary ammonium salt process for itspreparation. The invention further relates of N-benzyl heparinamidewhich is at least 90% amidified to novel anticoagulant compositionshaving an elevated and treating the latter with an aqueous solution ofsodium antilipemic and clarifying activity and to a novel method acetateto form the sodium salt of N-benzyl heparinamide of retarding thecoagulation of blood. which may be precipitated by the addition of alower alka- It is an object of the invention to provide the novel n01such as methanol. Other alkali metal salts of lower compound, N-benzylheparinamide, and its alkali metal alkanoic acids may be used in placeof sodium acetate. salts. As has been indicated above N-benzylheparinamide It is another object of the invention to provide a noveland its alkali metal salts are useful for the treatment or process forthe preparation of N-benzyl heparinamide and the prevention ofthrombosis, which can come on unexits alkali metal salts. pectedly incirculatory illnesses, phlebitis, arteritis, as

It is a further object of the invention to provide novel well as incardiac illnesses.

anticoagulant compositions having an elevated antilipemic Thesecompounds possess particularly the advantage and clarifying activity.over heparine of exercising a prolonged action which It is an additionalobject of the invention to provide avoids repeated intravenousinjections or continuous vea novel method of retarding the coagulationof blood. nous perfusions corresponding to very elevated doses of Theseand other objects and advantages of the invenheparine. They possess, inaddition, the advantage over tion will become obvious from the followingdetailed the anticoagulant derivatives of dicoumarine of actingdescription. with rapidity and during an easily determinable delay,

The novel products of the invention ar N-benzyl whereas the derivativesof dicoumarine which inhibit the heparinamide and its alkali metal saltswhich have the synthesis of thrombine in the liver (antagonists ofvitamin probable following structural formula K) have a delaying actionbut whose duration cannot be CONHCHzCaHg CHzOSOsR CONHGHQCGH5 CHzOSOsR Io o -o )-o r I 050311 OH J OH i k OH \l A L \1 \1 OH NHSOQR 3 0H NHSOQR11 wherein R is selected from the group consisting of hyforeseen withcertainty. Finally, for aught we know the drogen and an alkali metal.action of N-benzyl heparinamide and of its alkali metal Derivatives ofheparine having coagulating retarding salts is manifest on all stages ofcoagulation. This renactivity are known. However, N-benzyl heparinamidedcrs its action more sure and less dangerous for the orand its alkalimetal salts not only possess a prolonged ganism. anticoagulatingactivity, marked even at relatively small The products of the inventioncan be employed in gendoses, but also possess an elevated antilipemicand clarifyeral under all conditions of use of heparine with the adingactivity. vantage of a decrease in frequency of the injections.

The process of th inve tion for the re arati f The novel anticoagulantcompositions of the invention an alkali metal salt of N-benzylheparinamide comprises having an elevatfid iantilipemic and Clarifyingactivity are reacting a lower alkyl chloroformate with a high molecu-Comprised of a Compound Selected from the group C011- lar i ht long h iquaternary ammonium lt f sisting of N-benzyl heparinamide and its alkalimetal salts h i t f a i d h d id d reacting th and a major amount of apharmaceutical carrier. The latter with mono benzylamine to form thecorresponding Compositions y be in the form Of aqueous inle'ctablequaternary ammonium salt of N-benzyl heparinamide SOlullOl'LS andsuppositories prepared in the usual manner. which may be reacted with analkali metal salt of a lower The met'hfld 0f h 111Vent 10}1 fetflrdlng talkanoic acid to form the corresponding alkali metal coagulation ofblood COmPIISES admlnlsteflng an efiffct lve Salt f Nqjenzylheparinamide amount of a compoundselectedfrom the group consisting hprocess f h preparation f Nbenzy} heparim of N benzyl heparinamide and1t s alkali metal salts. The amide comprises reacting a lower alkylchloroformate Sald P P may be adffllnlstered rectally trailswith a highmolecular weight long chain quaternary cutaneously 1n the form ofintramuscular, subcutaneous monium salt f heparin to f the correspondingmixed or intravenous in ections. The usual useful dosage is beanhydrideand reacting the latter with monobenzylamine tween 100 d g- Per daydepending upon the to form the corresponding quaternary ammonium salt ofmethod of N-benzyl heparinamide which is reacted with an alkali In thefollowlflg pl there fiescrl'l 3ed SeVBTal metal salt of a lower alkanoicacid to form the preferred embodiments to illustrate theIIIlVfiIltltJl'l; Howspending alkali metal salt f Ngbenzyl heparinamide,the ever, it should be understood that the invention is not aqueousSolution of which is treated by an acid ion intended to be limited tothe specific embodiments. change resin to form N-benzyl heparinamide. XAL 1 The high molecular weight long chain quaternary ammonium compoundsused to form the corresponding salt of heparine are such as for examplebenzyldimethyl-2-[2- cc. of tetrahydrofuran and 10 gm. of the neutralPreparation of the sodium salt of N-benz yl heparinamide 3 Hyamine 1622salt of heparine (prepared according to U.S. Patent No. 2,989,438) wereintroduced into a threenecked balloon flask and the mixture was agitateduntil total dissolution occurred. Then, the mixture was cooled 'to and3.3 cc. of ethyl chloroformate were added. Agitation was continued andthen 25 cc. of monobenzylamine were added. A precipitate of N-benzylheparinamide occurred and the reaction mixture was held overnight in arefrigerator. Thereafter the reaction mixture was evaporated undervacuum and the residue was taken up in 100 cc. of butanol. T hebutanolic solution was extracted several times with an aqueous solutioncontaining 20% of sodium acetate. The sodium salt of N-benzylheparinamide was precipitated from methanol and the precipitate wasvacuum filtered and washed with methanol. Thereafter the precipitate wastaken up in distilled water and 300 cc. of a solution of Hyamine 1622were added thereto. The Hyamine salt precipitated and the salt wasseparated, washed with distilled water and dried under vacuum. Thebenzyl heparinamide was isolated thus in the form of its Hyamine 1622salt and weighed 9.5 gm.

One proceeds to a second amidification on the 9.5 gm. of productobtained above under the same conditions as that preceding. There wasfinally obtained 2.7 gm. of

benzyl heparinamide in the form of its sodium salt which had anamidification value of 93%.

'The sodium salt of N-benzyl heparinamide occurred in the form of anamorphous white powder and was very soluble in water, soluble in diluteaqueous acids and dilute aqueous alkalis and insoluble in alcohol,ether, acetone, benzene and chloroform. Its specific rotation was [a]=+69.8 (c.=1% in water).

Ultraviolet absorption corresponding to 1.3 benzyl groups per ram(theoretical: 1,4).

U./ mg. Anticoagulant activity in vitro 28 Antilipemic activity in vitro145 Analysis of the sodium salt of benzyl heparinamide, raw formulamolecular weight: (2,828.30),,.

Calculated: C, 32.27; H, 3.31%; N, 3.95; ash 27.60. Found: C, 32.7,32.9; H, 3.8, 3.9; N, 3.8, 3.8; ash, 26.85.

This compound is not described in the literature.

PHARMACOLOGICAL STUDY OF N-BENZYL HEP- ARINAMIDE AND ITS ALKALI METALSALTS (1) Immediate anticoagulant activity in vitro A plasma reactantdeprived of calcium and prothombine was prepared. By adding thrombine tothis plasma, the fibrinogen which it contains Was transformed intofibrin which produced simultaneously a coagulation and an opacification.The previous addition of an anticoagulant such as heparine provokes aretardation of the coagulation and of the opacification. If the timenecessary to obtain a given value of opacity is measured, it can beobserved that there exists a relation between this time and thecoagulating activity of the preparation. The values obtained arehereafter reported as those found compared to a solution of controlheparine. The values are expressed in antithrombic units (U.A.T.).

RESUDTS U.A.T./mg. Heparine control (sodium salt) 130140 The sodium saltof N-benzyl heparinamide 25-28 (2) Prolonged anticoagulating action invivo by intravenous method This action was studied in the rabbit bydetermining the time of coagulation at regular intrevals (2, 4, 6, 8 and10 hours after intravenous injection of the compound studied). Thecompounds were administered at doses of 2 10 mg./kg. and 20 mg./kg. Thefollowing results have been obtained:

As can be seen from this table, the sodium salt of N-benzyl heparinamidepossesses a prolonged anticoagulant action at anticoagulant doses(expressed in units) infinitely lower than those of heparine necessaryto obtain an effect of the same order.

(3) Clarifying activity in vitro The clarifying activity of the sodiumsalt of N-benzyl heparinamide was determined by utilizing the techniquepublished by Plotka and Jequier (Arch. Int. Pharmacodyn., 1960, 126,After having verified on several samples of heparine that the variationsof anticoagulant and antilipemic values were always parallel, a heparinetesting 150 anticoagulant units per mg. was taken as a referencestandard and was given the arbitrary value of 15 0 clarifying orantilipemic units which was used to define the antilipemic activity ofthe sodium salt of N-ben- Zyl heparinamide in antilipemic units. To thisefiect, the variations of turbidity caused by the action of increasingdoses of standard heparine were measured in the presence of CaCl on thesame volume of artificial substrate of egg yellow. In certain limits ofconcentration of heparine, the graphic representation was a straightline. The test was repeated with the sodium salt of N-benzylheparinamide and, with reference to the reference graph, the value inantilipemic units (A.L.) of the sample was found to be U./mg.

(4) Determination of toxicity The sodium salt of N-benzyl heparinamidewas placed in solution in physiological serum and administered to lotsof ten mice of the Rockland strain weighing between 18 and 22 gm. Thecompound was injected intravenously in a volume of 10 cc. per kg. ofanimal at doses, respectively, of 100 mg./ kg. and 200 mg./ kg. Theanimals held under observation for a period of one week manifested nosign of intoxication. There was no mortality observed at a dose of 200mg/kg. Therefore, the sodium salt of N-benzyl heparinamide in acutetesting on mice at a dose of 200 mg. /kg. administered intravenously isdevoid of toxicity.

Various modifications of the compositions and process of the inventionmay be made without departing from the spirit or scope thereof, and itis to be understood that the invention is to be limited only as definedin the appended claims.

I claim:

1. A compound selected from the group consisting of N-benzylheparinamide and its alkali metal salts.

2. N-benzyl heparinamide.

3. The sodium salt of N-benzyl heparinamide.

4. A process for the preparation of an alkali metal salt of N-benzylheparinamide which comprises reacting a lower alkyl chloroformate with ahigh molecular weight quaternary ammonium salt of heparine to form thecor-. responding mixed anhydride and reacting the latter withmonobenzylamine to form the corresponding quaternary ammonium salt ofN-benzyl heparinamide which is re;

acted with an alkali metal salt of a lower alkanoic acid to form thecorresponding alkali metal salt of N-benzyl heparinamide.

5. The process of claim 4 wherein the said alkali metal salt of a loweralkanoic acid is sodium acetate.

6. The process of claim 4 wherein the lower alkyl chloroformate is ethylchloroforrnate.

7. A process for the preparation of the sodium salt of N-benzylheparinamide which comprises reacting ethyl chloroformate with a highmolecular weight quaternary ammonium salt of heparine in the presence ofan inert organic solvent to form a mixed anhydride, reacting the latterwith monobenzylamine to form the corresponding quaternary ammonium saltof N-benzyl heparinamide and reacting the latter with an aqueoussolution of sodium acetate to form the sodium salt of N-benzylheparinamide.

8. A process for the preparation of N-benzyl heparinamide, whichcomprises reacting a lower alkyl chloroformate with a high molecularweight quaternary ammonium salt of heparine to form the correspondingmixed anhydride and reacting the latter with monobenzylamine to form thecorresponding quaternary am monium salt of N-benzyl heparinamide whichis reacted with an alkali metal salt of a lower alkanoic acid to formthe corresponding alkali metal salt of N-benzyl heparinamide, theaqueous solution of which is treated by an acid ion exchange resin toform N-benzyl heparinamide.

9. An anticoagulant composition comprising a compound selected from thegroup consisting of N-benzyl heparinamide and its alkali metal salts anda major amount of a pharmaceutical carrier.

10. The composition of claim 9 containing 100 to 600 mg. of theanticoagulating compound.

11. The composition of claim 9 wherein the anticoagulating compound isthe sodium salt of N-benzyl heparinamide.

No references cited.

JULIAN S. LEVITT, Primary Examiner.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF N-BENZYLHEPARINAMIDE AND ITS ALKALI METAL SALTS.